1. Technical Field
The present invention relates generally to capillary electrophoresis. More specifically, the invention relates to a novel method for carrying out capillary electrophoresis using zwitterion-coated capillary tubes. The use of zwitterionic coatings in turn provides for greater control over electroosmotic flow and minimizes adsorption of components contained within the sample solution. The invention additionally relates to an apparatus for performing the novel method.
2. Description of the Related Art
Separation of chemical entities is possible using the technique of "electrophoresis", a method which is premised upon the differential migration of solutes in an electric field. In high performance capillary electrophoresis (HPCE), a technique developed in the early 1980's, electrophoretic separation is performed in narrow capillary tubes, typically 25 .mu.m to 75 .mu.m in diameter, which are filled with a conducting solution, normally a buffer. A small amount of sample is introduced at one end of the capillary tube, followed by application of a high potential difference across the ends of the tube. Electroosmotic flow (also termed "electroendoosmotic flow", or EOF) and differences in electrophoretic mobilities combine to provide a spatial separation of the constituents of the sample solution.
HPCE has numerous advantages, particularly with respect to the detrimental effects of Joule heating. The high electrical resistance of the narrow capillary tube enables the application of very strong electric fields, in the range of 100 to 500 V/cm, with only minimal heat generation. Additionally, the large surface-to-volume ratio of the capillary enables efficient dissipation of the heat that is generated during the separation process. Further, the use of high electric fields gives rise to shorter analysis times (on the order of ten minutes or less) than required for conventional electrophoretic separations, high separation efficiency, and superior resolution. Peak efficiency, often in excess of 10.sup.5 theoretical plates, is due in part to the plug profile of the EOF, which also enables the simultaneous analysis of all solutes, regardless of charge. Finally, HPCE allows for use of relatively simple instrumentation, on-line detection through the capillary wall, and very small sample volumes (on the order of 1 to 10 nl).
The underlying principles of electrophoretic separation are quite simple, and may be summarized as follows. Separation of constituents in a sample is enabled based on differences in solute velocity in an electric field. The velocity of an ion is given by the relationship: EQU v=.mu..sub.e E
where v is the ion velocity, .mu..sub.e is the electrophoretic mobility, and E is the applied electric field. The electrophoretic mobility, for a given ion and medium, is a constant which is characteristic of that ion, and may be represented as: EQU .mu..sub.e =q.div.6.pi..sup.n r
where q is the ion charge, .sup.n is the solution viscosity, and r is the ion radius. From this relationship, it is evident that small, highly charged species have high mobilities whereas large, minimally charged species have low mobilities. It will be appreciated that the electrophoretic mobility found in standard tables is a physical constant, determined at the ideal conditions of full solute charge and infinite dilution, and differs somewhat from the electrophoretic mobility that is determined experimentally. The experimental value is termed the "effective mobility" and is highly dependent on the pH of the sample in the bulk fluid.
The pH of the sample undergoing analysis is important in another respect as well. The EOF velocity V.sub.EOF, may be defined as follows: EQU v.sub.EOF =(.epsilon..zeta./.sup.n)E
where .epsilon. is the dielectric constant of the sample fluid and .zeta. is the zeta potential (and .epsilon..zeta./.sup.n is the electroosmotic mobility, .mu..sub.EOF). The zeta potential .zeta. is essentially determined by the surface charge on the capillary wall, which is in turn related to the presence of the surface silanol groups on the interior of the capillary tube. These surface silanol groups are predominantly deprotonated at higher pH (such that they are in the form of anionic, Si--O.sup.- groups) and protonated at lower pH (such that they are in the form of ionically neutral Si--OH groups). The magnitude of the EOF velocity, then, is strongly pH dependent.
In some cases, a higher EOF velocity is preferred, i.e., when working with materials that are readily separated. In many other cases, however, a high V.sub.EOF can result in elution of solute before separation has occurred, and a lower EOF velocity is preferred. In still other cases, an EOF flow counter to the direction of ion migration is desirable. Accordingly, the ability to control EOF velocity is highly desirable. To date, however, electronic control of EOF velocity has been possible only for electrophoretic separations conducted at a very low pH.
The present invention is addressed to the aforementioned need in the art, as it is directed to a method for broadening the pH range within which EOF can be regulated, i.e., within which EOF velocity can be controlled. The invention is premised on the discovery that the "electric double layer" caused by the buildup of negative charges on the interior of the capillary tube may be controlled by coating the interior of the capillary with a zwitterionic species. In addition to broadening the pH range within which EOF velocity may be controlled, the zwitterionic coatings minimize adsorption of sample constituents to the capillary wall via ionic attraction to the ionized silanol groups, and thus enhance the efficiency and resolution of separation.